3',4'-Dideoxykanamycin B which was synthetically derived by us from kanamycin B (refer to Japanese Patent Publications Nos. 7595/75 and 46110/76; U.S. Pat. No. 3,753,973) has been used widely as a chemotherapeutic agent characterized by its high activity against kanamycin-resistant microbial strains including Pseudomonas aeruginosa. More recently, 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin B (refer to Japanese Patent Publication No. 33629/77; U.S. Pat. No. 4,107,424) and 1-N-isoseryl-3',4'-dideoxykanamycin B (refer to Japanese Patent Prepublication No. 47949/75; U.S. Pat. No. 3,939,143) were synthesized also by us and found to exhibit a high activity against a wide variety of drug-resistant microbial strains including Pseudomonas aeruginosa. Our continued studies of these kanamycin derivatives have now resulted in the development of a new, convenient route for large-scale preparation of 1-N-isoseryl- or 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-dideoxykanamycin B.